Treatment of viruses



United States Patent 3,259,546 TREATMENT OF VIRUSES John R. Polley,Ottawa, Ontario, Canada, assignor to Canadian Patents and DevelopmentLimited, Ottawa, Ontario, Canada, a company of Canada No Drawing. FiledJune 1, 1962, Ser. No. 199,250 4 Claims. (Cl. 16778) This inventionrelates to the preparation of vaccines suitable for immunization againstvirus infections. Virus suspensions containing certain protective agentsare subjected to controlled doses of ionizing radiation.

Vaccines have been prepared from infective viruses by chemicalinactivation using formaldehyde, ethylene oxide, fi-propiolactone, orsimilar compounds. A combination of this chemical treatment withultra-violet or ultra-sonic irradiation has been used. Inactivation withultra-violet, ultra-sonic, high speed electron or gamma radiation haspreviously been investigated. By the above treatments it has beendifficult in practice to consistently arrive at complete destruction ofthe infectivity'while retaining a large percentage of the antigenicity.Treatments with ionizing radiation in the .past gave unpredictableresults-the complete inactivation was not consistently related to theradiation dose, and the antigenicity was often destroyed at an equal orgreater rate than the infectivity. When the infectivity has not beencompletely destroyed by the initial treatment it has been practicallyimpossible to re-treat without also destroying a significant amount ofthe antigenicity. Wastage of batches in the preparation of some vaccinesfrom live virus, has been significant. The prior methods have not madefeasible the preparation of vaccines from all types of viruses.

An object of this invention is to prepare vaccines from various liveviruses by completely destroying the infectivity While consistentlyretaining the antigenicity. Another object is to provide a controlledprocess for preparing vaccine from live virus, utilizing ionizingradiation. A further object is to protect the antigenicity of the virusduring irradiation. A still further object is to provide a viruscomposition containing certain protective agents for the antigenicity.

It has now been found that it is possible to apply an additionalcalculated dose of ionizing radiation to destroy any residualinfectivity remaining after the initial irradiation. It has now beenfound according to the invention that, in the presence of certainchemicals, the rate of destruction of antigenicity on irradiation issignificantly, and consistently, lower than the rate of destruction ofthe infectivity. The radiation dose can be controlled to just completelydestroy the infectivity and yet consistently retain the antigenicity.The permissible excess over the required dose, before the antigenicityis significantly lowered, is broad enough to allow effective control incommercial practice.

Any ionizing radiation, such as X, y, 3, or electron radiation, may beused. It is presently preferred to use gamma radiation such as thatproduced by a commercially available cobalt-60 irradiator. The radiationdose (to completely destroy the infectivity) is usually within the rangefrom about 0.5 to about 6x10 rads, depending on the particular virus,virus concentration, protective agent, and protective agentconcentration used.

The protective agents desirably are water soluble to an effectiveconcentration, easy to handle, and have no undesirable effect on thevirus antigenicity. Also the agents desirably have no seriousphysiological toxicity 3,259,546 Ice Patented July 5, 1966 The additiveswhich have been found to protect the antigenicity in preference to theinfectivity are compounds which also are antioxidant in nature, orpreferably combine with nascent oxygen or oxidizing agents produced inthe system. Alternatively the additives may act primarily as freeradical scavengers, or be effected preferentially by free radicals, andselectively transmit the energy involved. The compounds should be noteasily decomposed, and preferably capable of resonance or existance indifferent tautomeric forms. Suitable compounds include certainsulphur-containing amino-acids, aromatic ring compounds having at leasttwo polar substituent groups, heterocyclic compounds havingunsaturation, unsaturated hydroxy acids and mixtures thereof.Illustrative compounds are cystine, histidine, tryptophan, tyrosine,allantoin, phenylalanine, alkali metal p-aminohippurate, sulphanilamide,and ascorbic acid. The free acids or nonstoxic salts of the variouscarboxylic acids may be used provided that the pH of the system ismaintained above about 5. Aniline and pyridine exert a pro tectiveaction but are physiologically undesirable. Cysteine and methionineproduced unpleasant odours suggesting the rupture of carbon-sulphurbonds, but are otherwise good protective agents. Histidine and sodiumpamino-hippurate are presently preferred for influenza and mumps virusesrespectively.

The concentration of the protective agent in the virus suspension mayrange from about 0.01 to about 1% (wt./vol.). As the concentration isincreased the radiation dose required to destroy the infectivityincreases. The concentration limits are not critical and some variationabove or below the given limits may be tolerated. The preferredconcentration is constant and within the range 0.05 to 0.3%. If theconcentration used is too high, the radiation dose required will beexcessive.

The suspension of the live virus is obtained in any suitable manner. Itmay be desirable to purify the initial suspension if impurities arepresent which will cause a nonuniform unpredictable radiationdose-infectivity relationship. Suspensions of the larger viruses arereadily purified by oentrifugation and resuspension. Other methods areknown to those skilled in the art. In some instances an unpurifiedsuspension in selected tissue culture medium can be used directly. Themedia for these suspensions are usually aqueous saline solutions orliquids suitable for inoculation. For control and optimum efficiency ofthe process it has been found desirable to provide a selectedstandardized protective agent system for each virus, giving a knowninfectivityzradiation dose curve (and allowing ready calculation of anysupplementary dose required). This may involve the use of a selectedculture medium, or purification of the virus-containing suspensionfollowed by addition of effective amounts of a selected agent orsynergistic combinations thereof. These steps may be done singly or incombination.

The protective action of various chemical additives on the antigenicityof influenza A virus is illustrated. Virus suspensions were prepared bycentrifuging freshly harvested allantoic fluid containing influenza A(PR8) virus at 10,000 rpm. for one hour (8700 g.). The sediment wasresuspended in saline to produce a hemagglutination titer of about1:2560 per ml. In a few instances initial titers of about 1:5120 or1:10,240 were used. However, the decreased titers after irradiationrapidly become similar as the dose is increased.

For irradiation 10 ml. samples of these suspensions were placed in smallglass vials with plastic screw caps. The vials were irradiated in acobalt=60 Gammacell 220 irradiator (Commercial Products Division, AtomicEnergy of Canada Limited) at a dose rate of 1.25 X 10 rads/ hour. Aftervarious exposure times the vials were stored overnight at 4 C. and thentested for infectivity and hemagglutination titer (as described in Can.J. Microbiol, 1, 25 6- 61, 1955). The hemagglutination titer is taken asa good measure of the antigenicity for these pneumotropic viruses. Atiter of greater than about 1:300 is usually necessary for humanvaccines. Other tests may be used for the antigenicity, includingobservation of inoculated animals, as are known in the art.

Example I infectivity is increased. At the lower concentrations of PAHthe hemagglutinin was significantly decreased before the infectivity wasdestroyed. The histidine was effective over the range of concentrationsshown and permitted considerable excess radiation before thehcmagglutinin was seriously affected. About 0.1% histidine is preferredfor this influenza virus. The PAH has been found to be more advantageouswith mumps virus. The ratio of the radiation dose completely removingthe infectivity, to the removing the hemagglutinin, for thehistidineinfluenza A system, is about 0.2. In general this ratio shouldbe less than about 0.7.

The infectivity and antigenicity decrease approximately exponentiallywith increase in radiation dose, for most viruses. The infectivity canbe reduced to approximately the same extent regardless of whether theradiation is administered as a single dose or in divided doses. From theinitial rate of the virus inactivation it is possible to TABLEI.INFLUENZA A VIRUSHEMAGGLUTININ UNITS PER ML.

Radiation Dose (rads) Reagent Added 0 (Control) 2, 560 20 20 20 20Cystine 2, 560 2, 560 2, 560 640 160 20 Cysteine 2, 560 2, 560 2, 560 2,560 320 160 Methionine 5, 120 5, 120 2, 560 2, 560 1, 280 320 Histidine2, 560 2, 560 2, 500 2, 560 1, 280 160 Tryptophan 2, 560 2, 560 1, 280320 20 20 Tyrosine 5, 120 2, 560 2, 560 320 20 20 2, 560 1, 280 640 2020 20 5, 120 2, 560 1, 280 040 80 20 Sodium p-aininohippurate 2, 560 2,560 2, 560 640 320 40 Sulphanilamide 5, 120 2, 560 1, 280 640 80 20Ascorbic Acid 2, 500 2, 560 1, 280 320 20 20 Aniline 2, 560 2, 560 2,560 640 20 20 Pyridine- 2, 500 1, 280 1, 280 160 20 20 Calf Serum 5, 1202, 560 320 20 20 20 Bovine Albumin 2, 560 2, 560 320 20 20 20 2, 560 1,280 160 20 20 20 2, 560 160 20 20 20 20 2, 560 160 20 20 20 20 2, 560160 20 20 20 20 2, 560 80 20 20 20 20 2, 560 1, 280 160 20 20 20 2, 5602, 560 160 20 20 20 Arg'inine 2, 560 1, 280 20 20 20 20 Glycine 2, 500640 20 20 20 20 Urea 2, 560 1, 280 160 20 20 20 Quinhydrone 2, 560 20 2020 20 20 Allyl Alcohol 2, 560 640 320 20 20 20 In Table I, the compoundsbelow ascorbic acid are not suitable protective agents for thehemagglutinin. Phenylalanine ofiered protection up to 1X10 rads andcould be used in some instances. Carbohydrates such as glucose, sucrose,and inulin had little effect. Proteins such as calf serum and bovinealbumin afiorded only a small measure of protection. Simplestraight-chain amino acids, not containing sulphur, such as glycine andarginine had a relatively small effect.

Example 11 The hemagglutinin and infectivity of influenza A virus wasmeasured for various gamma radiation doses and for variousconcentrations of histidine and sodium p-aminohippurate (PAH). Theresults are shown in Table II.

calculate the radiation dose required to destroy the infectivity. If agiven dose has been insuflicient the suspension can be subjected to afurther dose, the amount of which can be exactly calculated from theknown complete in activation curve for the system involved. Theinactivation commences and ceases almost simultaneously with thecommencement and cessation of irradiation. The rate of irradiation ordose rate is of minor importancethe total dose being most significant.

The mechanism of the inactivation is not understood but is believed tobe largely an indirect chemical effect and not a primary result of theradiation per se. It may be that free radicals produced transfer theirenergy via chemical intermediaries exerting an oxidizing action. The

TABLE II.INFLUENZA A VIRUS-HEMAGGLUTININ UNITS PER ML.

Iiistidino Concentration PAH Concentration Radiation Control Dose (rods)0 5, 5, 120 5, 120 5, 120 5, 120 5, 120 10, 240 025x10 2,560 5,120 5,1205, 120 5, 120 5, 120 10, 240 0.5 10 1 1 5, 120 5, 120 5, 120 5, 120 5,120 10, 240 1.0X10 1 0 1 2, 560 1 5, 120 5, 120 1 2, 560 5, 120 10, 2402.0X 10 1 1, 280 1 2, 560 1 5, 120 1 640 1 1, 280 1 10,240 4.0 10 1 40 1320 1 1, 280 1 40 1 160 1 l, 280 6.0)(10 20 20 1 80 20 20 20 1 The virussuspension was non-infective at this dose and concentration.

From Table II it is evident that as the concentration protective agentsof the invention appear to counteract the is increased the dose requiredto completely destroy the 75 oxidizing action or intercept the energytransfer from free radicals or both, preferentially for the antigeniccomponent.

The process of this invention may be used to prepare vaccines fromvarious viruses. Suitable protective agents, type of ionizing radiationand radiation dose will be readily ascertainable by one skilled in theart from the above teachings. The above examples are illustrative onlyand are not intended to limit the invention which is defined in theappended claims.

I claim:

1. A process for destroying infectivity by irradiating aqueoussuspensions of infective virus selected from the group consisting ofinfluenza and mumps, comprising adding to the suspension a protectiveagent for the virus antigenic component selected from the groupconsisting of cystine, cysteine, methionine, histidine, tryptophan,tyrosine, phenylalanine, allantoin, p-aminohippuric acid,sulphanilamide, ascorbic acid and non-toxic salts of the acids, in aneffective amount from about 0.05 to about 0.08% wt./vol., irradiatingwith gamma radiation until the infectivity is destroyed, the radiationdose 'being within the range 1 10 to 6x10 rads, and recovering virussuspensions having a hemagglutinin (units/ml.) of at least about 300,and suitable for vaccine preparation.

2. A process for destroying infectivity by irradiating aqueoussuspensions of infective mumps virus, comprising adding to thesuspension a protective agent for the mumps virus antigenic componentconsisting essentially of sodium p-aminohippurate in an eflective amountfrom about 0.05 to 0.8% wt./vol., irradiating with gamma radiation untilthe infectivity is destroyed, the radiation dose being within the range1X10 to 6 10 rads and recovering suspensions having a hemagglutinin(units/ ml.) of at least about 300 and suitable for vaccine preparation.

3. A process for destroying infectivity by irradiating aqueoussuspensions of infective influenza virus, comprising adding to thesuspension a protective agent for the influenza virus antigeniccomponent consisting essentially of histidine, in an effective amountfrom about 0.05 to 0.8% wt./vol., irradiating with gamma radiation untilthe infectivity is destroyed, the radiation dose being within the range1 10 to 6 10 rads, and recovering suspensions having a hemagglutinin(units/ml.) of at least about 300 and suitable for vaccine preparation.

4. A process for destroying infectivity by irradiating purified aqueoussaline suspensions of infective influenza A (PR8) virus, comprisingadding to the suspension a protective agent for the influenza virusantigenic component consisting essentially of histidine, in an eflectiveamount from about 0.1 to 0.3% wt./vol., irradiating with gamma radiationuntil the infectivity is destroyed, the radiation dose being Within therange 1 10 to 4 10 rads, and recovering suspensions having ahemagglutinin (units/ml.) of at least about 300 and suitable for vaccinepreparation.

References Cited by the Examiner UNITED STATES PATENTS 2,421,382 6/1947Levinson et al. 167-78 2,445,301 7/1948 Chambers 167-78 3,019,168 1/1962Taylor 167-78 3,031,378 4/1962 =Ishidate et al. 167-78 3,060,094 10/1962Dutcher et al 167-78 3,061,518 10/1962 Averswald et al. 167-78 3,065,13911/1962 Ericsson 167-72 3,076,748 2/1963 Lo Grippo et al 167-783,105,011 9/ 1963 McLean et al. 167-78 OTHER REFERENCES Jordan et al.,Inactivation of Some Animal Viruses With Gamma Radiation From Cobalt-60,Proc. Soc. Exp. Biol. Med., 91(2): 212-215, February 1956.

Rivers et al., Viral and Rickettsial Infections of Man, 3rd Ed., pp.32-34, 39-42, -112, 162-168, 215-216, 224-226, 239-246, 502-504,575-576, 643-646, 783, 786-787, published 1959 by J. B. Lippincott,Philadelphia, Pa.

LEWIS GO'ITS, Primary Examiner.

S. K. ROSE, Assistant Examiner.

1. A PROCESS FOR DESTROYING INFECTIVITY BY IRRADIATING AQUEOUSSUSPENSIONS OF INFECTIVE VIRUS SELECTED FROM THE GROUP CONSISTING OFINFLUENZA AND MUMPS, COMPRISING ADDING TO THE SUSPENSION A PROTECTIVEAGENT FOR THE VIRUS ANTIGENIC COMPONENT SELECTED FROM THE GROUPCONSISTING OF CYSTINE, CYSTEINE, METHIONINE, HISTIDINE, TRYPTOPHAN,TYROSINE, PHENYLALANINE, ALLANTON, P-AMINOHIPPURIC ACID, SULPHANILAMIDE,ASCORBIC ACID AND NON-TOXIC SALTS OF THE ACIDS, IN AN EFFECTIVE AMOUNTFROM FROM ABOUT 0.05 TO ABOUT 0.08% WT./VOL., IRRADIATING AND GRAMMARADIATION UNTIL THE INFECTIVITY IS DESTROYED, THE RADIATION DISE BEINGWITHIN THE RANGE 1X10**6 TO 6X10**6 RADS, AND RECOVERING VIRUSSUSPENSIONS HAVING A HEMAGGLUTININ (UNITS/ML.) OF AT LEAST ABOUT 300,AND SUITABLE FOR VACCINE PREPARATION.